Carboxyl terminal domain basic amino acids of mycobacterial topoisomerase I bind DNA to promote strand passage

Bacterial DNA topoisomerase I (topoI) carries out relaxation of negatively supercoiled DNA through a series of orchestrated steps, DNA binding, cleavage, strand passage and religation. The N-terminal domain (NTD) of the type IA topoisomerases harbor DNA cleavage and religation activities, but the carboxyl terminal domain (CTD) is highly diverse. Most of these enzymes contain a varied number of Zn2+ finger motifs in the CTD. The Zn2+ finger motifs were found to be essential in Escherichia coli topoI but dispensable in the Thermotoga maritima enzyme. Although, the CTD of mycobacterial topoI lacks Zn2+ fingers, it is indispensable for the DNA relaxation activity of the enzyme. The divergent CTD harbors three stretches of basic amino acids needed for the strand passage step of the reaction as demonstrated by a new assay. We also show that the basic amino acids constitute an independent DNA-binding site apart from the NTD and assist the simultaneous binding of two molecules of DNA to the enzyme, as required during the catalytic step. Although the NTD binds to DNA in a site-specific fashion to carry out DNA cleavage and religation, the basic residues in CTD bind to non-scissile DNA in a sequence-independent manner to promote the crucial strand passage step during DNA relaxation. The loss of Zn2+ fingers from the mycobacterial topoI could be associated with Zn2+ export and homeostasis.

Type IA topoisomerase inhibition by clamp closure

Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.

Conditional silencing of topoisomerase I gene of Mycobacterium tuberculosis validates its essentiality for cell survival

Topoisomerases are an important class of enzymes for regulating the DNA transaction processes. Mycobacterium tuberculosis (Mtb) is one of the most formidable pathogens also posing serious challenges for therapeutic interventions. The organism contains only one type IA topoisomerase (Rv3646c), offering an opportunity to test its potential as a candidate drug target. To validate the essentiality of M.tuberculosis topoisomerase I (TopoI(Mt)) for bacterial growth and survival, we have generated a conditionally regulated strain of topoI in Mtb. The conditional knockdown mutant exhibited delayed growth on agar plate. In liquid culture, the growth was drastically impaired when TopoI expression was suppressed. Additionally, novobiocin and isoniazid showed enhanced inhibitory potential against the conditional mutant. Analysis of the nucleoid revealed its altered architecture upon TopoI depletion. These studies establish the essentiality of TopoI for the M.tuberculosis growth and open up new avenues for targeting the enzyme.

Direct regulation of topoisomerase activity by a nucleoid-associated protein

The topological homeostasis of bacterial chromosomes is maintained by the balance between compaction and the topological organization of genomes. Two classes of proteins play major roles in chromosome organization: the nucleoid-associated proteins (NAPs) and topoisomerases. The NAPs bind DNA to compact the chromosome, whereas topoisomerases catalytically remove or introduce supercoils into the genome. We demonstrate that HU, a major NAP of Mycobacterium tuberculosis specifically stimulates the DNA relaxation ability of mycobacterial topoisomerase I (TopoI) at lower concentrations but interferes at higher concentrations. A direct physical interaction between M. tuberculosis HU (MtHU) and TopoI is necessary for enhancing enzyme activity both in vitro and in vivo. The interaction is between the amino terminal domain of MtHU and the carboxyl terminal domain of TopoI. Binding of MtHU did not affect the two catalytic trans-esterification steps but enhanced the DNA strand passage, requisite for the completion of DNA relaxation, a new mechanism for the regulation of topoisomerase activity. An interaction-deficient mutant of MtHU was compromised in enhancing the strand passage activity. The species-specific physical and functional cooperation between MtHU and TopoI may be the key to achieve the DNA relaxation levels needed to maintain the optimal superhelical density of mycobacterial genomes.

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and Mycobacterium tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the Mycobacterium tuberculosis and Mycobacterium smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

Autoregulation of topoisomerase I expression by supercoiling sensitive transcription

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Es-cherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobac-terium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of suboptimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.

Mulier virilis. la masculinización del modelo femenino en el cristianismo primitivo: de Tecla a Melania Junior

131 p. - E-mail del autor: ikmagro88@gmail.com

O mundo, e suas máquinas: um estudo sobre propagação temática em A Máquina do Mundo, de Carlos Drummond de Andrade

Este estudo parte de preocupações filosóficas, teóricas e analíticas para promover exercícios de leitura e de interpretação do poema A Máquina do Mundo (1951), de Carlos Drummond de Andrade. Estabelecendo pela via ensaística a noção de ontologia da propagação, busca localizar a necessidade da ontologia no fundamento pré-paradigmático da ciência. Incorporando referências da Fenomenologia e da Hermenêutica, tenta assumir seus pressupostos e implicações para a constituição do método em Teoria da Literatura e para a definição dos campos básicos para a analítica da existência lógica, empírica e pragmática: os campos que representam as instâncias ontológicas do real, do simbólico e do imaginário. Da assunção fenomenológica e hermenêutica, passa-se a considerações sobre categorias pertencentes ao jargão literário, escolhidas por sua relação com o artefato literário em questão e correspondendo ao âmbito das três instâncias ontológicas: discute-se a Poesia como sendo um fenômeno constituinte, a Literatura como uma manifestação instituinte e o Poema como uma manifestação restituinte do signo literário A Máquina do Mundo. Em seguida, considera afetações e interferências de algumas correntes sociológicas, formalistas e antropológicas, buscando participar do diálogo sobre a possibilidade de aceitação da prática literária como uma prática de valor cognitivo, e não apenas ideológico e estético. Em seu terceiro momento, o estudo busca aplicar os pressupostos ontológicos e epistêmicos para estabelecer o limite dos espaços comparativos tornados possíveis ao poema A Máquina do Mundo e buscando o pano de fundo histórico e historiográfico do século XX a partir de suas possibilidades de relação com a obra de Drummond e segundo a orientação dos vetores dados no poema: a consolidação do veio crepuscular na tópica canônica do Novecentos brasileiro; a implicação da postura ideológica de Drummond na recepção crítica à sua obra; e, por fim, a localização posterior dos topoi cosmológicos e existenciais. Em seu último ensaio, refere-se à questão cosmológica e sua afetação sobre a postura existencial do Caminhante. Recuperam-se pontos de semelhança e de diferença entre as poéticas encontradas em Os Lusíadas, de Luís de Camões e em A Divina Comédia, de Dante Alighieri, para por fim remeter à identificação de uma continuidade temática entre o poema de Drummond e aquele que se costuma designar como pensamento originário na tradição da Filosofia ocidental. A seguir, buscam-se referências para alguns dos elementos formais do poema A Máquina do Mundo segundo sua origem no contexto da Literatura Ocidental. Consideram-se alguns traços característicos dos três seres representados (A Máquina, o Mundo, o Caminhante) e, encerrando-se a tese, procede-se a um exercício de leitura centrado especificamente no poema

Medieval Irish vision literature: a genre study

This dissertation examines medieval literary accounts of visions of the afterlife with an origin or provenance in Ireland from the perspective of genre, analysing their structural and literary characteristics both synchronically and diachronically. To this end, I have developed a new typology of medieval vision literature. I address the question in what manner the internationally attested genre of vision literature is adapted and developed in an Irish literary milieu. I explore this central research question through an interrogation of the typological unity of the key texts, both in formal arrangement and in the eschatological themes they express. My analysis of the structure and rhetoric of these narratives reveals the primary role of identity strategies, question-and-answer patterns and exhortation for their narrative cohesion and didactic function. In addition, I was able to make a formal distinction at text-level between the adaptation of the genre as an autonomous unit and the adaptation of thematic motifs as topoi. This further enabled me to nuance the distribution of characteristic features in the genre. My analysis of the spatial and temporal aspects of the eschatological journey confirms a preoccupation with personal eschatology. It reveals a close connection between the development of the aspects of graded access and trial in the genre and a growing awareness of an interim state of the soul after death. Finally, my dissertation provides new editions, translations and analyses of primary sources. My research breaks new ground in the hitherto underexplored area of genre adaptation in Ireland. In addition, it contributes significantly to our understanding of the nature of vision literature both in an Irish and a European context, and to our knowledge of the transmission of eschatological thought in the Latin West. Discusses the visions of: Laisrén, Fursa, Adomnán, Lóchán, Tnugdal, Owein and Visio Sancti Pauli Redactions VI and XI.

O legado clássico em Bocage: a elegia erótica latina e os sonetos amorosos bocageanos

O presente trabalho propõe-se analisar a influência da elegia erótica latina na produção poética de Bocage, nomeadamente nos sonetos amorosos, independentemente de se tratar de uma influência directa ou indirecta. Centra-se essencialmente em dois grandes aspectos: por um lado, na figura da mulher amada, traçando o seu retrato físico e psicológico e evidenciando a sua importância tanto na vida como na produção poética dos vates; por outro, analisam-se os tópicos do discurso amoroso, desde o momento do triunfo do amor sobre o sujeito poético até ao desejo de perpetuar esse amor para além da morte. Relativamente a estes dois aspectos, evidenciam-se semelhanças e diferenças no tratamento dos mesmos topoi. Contribuir-se-á, assim, para a valorização da formação clássica de Bocage, inúmeras vezes negligenciada pelos críticos.

O fantástico em Fialho de Almeida e Jean Lorrain pessimismo e decadentismo finisseculares

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Doutor em Estudos Portugueses - Estudos Comparatistas

Virtuosismo para instrumentário de sopro em Lisboa (1821-1870)

Dissertação apresentada para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Ciências Musicais

Aspects de la représentation de l'autre dans les romans grecs et "Les Métamorphoses" d'Apulée

Les romans grecs et les Métamorphoses d'Apulée - même si les modalités sont différentes pour ce dernier - sont des fictions en prose qui fonctionnent autour de topoi auxquels la figure de l'Autre n'échappe pas. Bien que le monde grec soit alors radicalement différent de ce qu'il était au Ve siècle avant J.-C, période à laquelle l'identité grecque est construite par opposition à la figure du barbare, les romanciers qui prennent la plume à partir du 1er siècle avant notre ère utilisent un certain nombre de stéréotypes hérités de l'époque classique, alors mise à l'honneur par le mouvement de la Seconde Sophistique. Il s'agit d'étudier dans le détail certains éléments de la représentation de l'Autre pour déterminer qui il est, comment il se comporte, ce qui le constitue en Autre. Puis, à partir de cette esquisse, nécessairement incomplète, d'évaluer ce que cette représentation peut induire sur l'image de l'identité grecque à l'époque impériale, par le jeu de miroir que F. Hartog a décelé dans l'oeuvre d'Hérodote. Une première partie est consacrée aux rapports entre l'homme et l'animal ainsi qu'à l'image de la sauvagerie, ce qui permet d'explorer les bornes romanesques de l'humanité. La seconde partie s'attache à des éléments que l'époque classique a plus particulièrement mis en avant pour distinguer les Grecs des non-Grecs : le critère de la langue, l'art de faire la guerre et le discours politique qui est tenu sur les institutions barbares. La troisième partie étudie la place des dieux et des pratiques religieuses dans la définition de l'Autre. J'espère ainsi contribuer à la compréhension du genre romanesque et des représentations culturelles de l'empire « gréco-romain ». -- The Greek novels and The Metamorphoses of Apuleius, even if it is in different terms for the last, are prose fictions which are based on topoi, and the figure of the Other is one of them. Although the Greek world was radically different of what it was in the fifth century BC, time during which Greek identity is contructed as opposed to the figure of the barbaros, the authors of novels, who wrote from the first century BC onward, used some stereotypes inherited from classical period, which was celebrated by the Second Sophistic movement. The aim of this thesis is to study in detail some elements of the representation of the Other to determine who it is, how he behaves, what makes him other. Then, from this sketch, necessarily incomplete, to evaluate what this representation says about the image of Greek identity in the imperial age, according to the play of the mirror detected by F. Hartog in the text of Herodotus. The first part of the thesis is dedicated to the relationship between man and animal and to the image of savagery, in order to explore the novelistic limits of humanity. The second part concentrates on elements that classical period had particularly insisted on to promote the distinction between Greeks and non-Greeks : the linguistic criterion, the way to make war, and the politic discourse on the barbaric institutions. The third part study the place of the gods and of religious practices in the definition of the Other. I hope to contribute to the understanding of novel genre and of cultural representations of the « greco- roman- empire ».